The present invention relates to the regulation of TNF-α expression by transcription factors that bind to a TNF-α promoter nucleotide sequence.
Tumor necrosis factor alpha (TNF-α) is a pleiotropic cytokine that is mainly produced by cells of the monocyte/macrophage lineage. TNF-α was originally identified as an endogenous factor, induced in response to inflammatory stimuli. Many studies have revealed that TNF-α exhibits both beneficial and pathologic effects and point to the importance of controlling the expression of this cytokine. For example, TNF-α and TNF-α-induced factors are known to contribute to the pathogenesis of inflammatory disorders (Alexander et al., J. Clin. Invest. 88:34-39, 1991; Sugarman et al., Science. 22:943-945, 1985; Beutler & Cerami, Annu. Rev. Immunol. 7:625-655, 1989; Talmadge et al., Cancer Res. 48:544-550, 1988; and Uglialoro et al., Tissue Antigens 52:359-367, 1998). The regulation of TNF-α gene expression in cells of the monocytic lineage is stimulus-dependent and quite complex, involving controls at both transcriptional and post-transcriptional levels. Many studies on the transcriptional regulation of TNF-α have focused on the investigation of transcription factors that bind to the responsive element sites within the TNF-α promoter, such as nuclear factor kappa B (NF-κB), Ets, NF-AT, activating protein-1 (AP1), cAMP response element binding protein (C/EBP) LPS-induced TNF-α factor (LITAF) and signal transducers and activators of transcription (STAT1). However, the relative contributions of these various regulatory elements in transcriptional activation of the TNF-α gene in human monocytes are poorly known.
Lipopolysaccharide (LPS), extracted from the outer membrane of Gram-negative bacteria, has been identified as a principal endotoxic component. LPS is a potent stimulator of monocytes and macrophages, inducing production and secretion of TNF-α and other inflammatory. The effects of LPS on transcription factor activity and expression have been widely investigated. Previous studies suggested that in vivo, LPS up-regulates the DNA binding activity of inducible transcription factors NF-κB, AP-1, and C/EBP in a time-dependent manner, but it down-regulates the DNA binding activity of constitutive transcription factors Sp1 and AP-2. The human TNF-α (hTNF-α) promoter contains motifs that resemble NF-kB-binding sites; however, controversy exists as to the involvement of NF-kB in TNF-α gene regulation. These sequences do not seem to be necessary for virus or LPS induction, nor do they appear to be able to stimulate virus or LPS induction alone. However, it has been suggested that NF-kB is an important factor in TNF-α gene transcription in LPS-challenged monocytes and macrophages. NF-kB-binding motifs are found in the hTNF-α promoter region and were shown to translocate into the nuclei of LPS-stimulated monocytes. In mice, mutation(s) or deletion(s) of NF-kB-binding motifs on the TNF-α promoter failed to show reporter gene activation in transfected cells. However, in humans, TNF-α promoter activity in transfected cell lines was found to be independent of the NF-kB-binding motifs. Drouet et al., J. Immunol. 147:1694-1700, 1991, offered an explanation for these conflicting data, suggesting that enough NF-κB is constitutively expressed to sustain high-level baseline expression of the human TNF-α gene compared with the mouse. An alternative explanation is that there may be another transcription factor acting independently or in concert with NF-κB in the activation of hTNF-α transcription.
In U.S. Pat. No. 6,566,501, such a factor is described. This polypeptide, termed liposaccharide-induced TNF-α factor (LITAF), was found to bind to the DNA-binding domain located from −550 to −487 in the promoter region of the human TNF-α gene. Furthermore, inhibition of human LITAF (hLITAF) mRNA expression in THP-1 cells resulted in a reduction of hTNF-α transcripts. It was also found that high levels of hLITAF mRNA are expressed predominantly in the placenta, peripheral blood leukocytes, lymph nodes, and the spleen.